The Wiskott Aldrich syndrome (WAS), an X-linked primary immunodeficiency characterized by deficient T cell function, thrombocytopenia and eczema, is caused by mutations in the WASP gene. WASP is recruited to the immune synapse (IS) where it is thought to be activated by the small GTPase Cdc42 to initiate Arp2/3 mediated actin polymerization. Actin polymerization is important for formation, IL-2 secretion and proliferation by T cells in response to TCR ligation. T cells from patients and mice deficient in WASP fail to increase their cellular F-actin, secrete IL-2 and proliferate followinq TCR ligation. This suggests that WASP plays a critical role in actin dependent changes that are essential for T cell activation. Our preliminary data suggests that following TCR ligation WASP can be recruited to lipid rafts and the IS by phosphorylated ZAP-70 and that this is mediated by the adapter protein CrkL which bridges p-ZAP-70 to the WASP partner WIP. Interestingly the majority of WASP missense point mutations in WAS are located in the WIP binding WH1 domain. Furthermore, there is data to suggest that the proline rich region of WASP which interacts with SH3 domain containing protein may also be involved in localization of WASP to the IS. We hypothesize that WASP recruitment to GEMs and the IS is mediated by WIP and by the polyproline rich region of WAS, and that it plays a critical role in T cell activation and in the dynamic actin changes in T cells following TCR ligation. To test this hypothesis, we propose to: 1. Analyze the ability of WASP-/- T cells to form an immune synapse and to undergo actin based cytoskeletal changes following TCR ligation by a). examining IS formation with anti-CD3 coated beads, MHC class II-peptide bilayers and antigen presenting cells, b). analyzing reorganization of cellular F-actin, actin cytoskeleton architecture changes and cell motitity following engagement of the TCR/CD3 complex. 2. Examine the ability of WASP-/- T cells to form lipid rafts in which key signaling molecules are concentrated leading to activation of the IL-2 gene. We will analyze the composition and stability of lipid rafts and the sustained activation of Ca ++mobilization, signaling intermediates and transcription factors that regulate IL-2 gene expression. 3. Assess the role of WIP binding and of the polyproline rich region of WASP in WASP recruitment to lipid rafts and IS. We will generate and test WASP mutants that fail to bind WIP or that lack the polyproline rich region, and examine the recruitment to lipid rafts and IS of WASP mutants that fail to bind WlP or lack the polyproline rich region. 4. Dissect the role of WASP recruitment to lipid rafts in TCR signaling. We will analyze TCR signaling in T cells of WASP-/- mice reconstituted with WASP mutants that perturb its recruitment to lipid rafts. We will determine if targeting signals in WASP mutants to lipid raft bypasses the physiologic pathway of WASP recruitment to lipid rafts for TCR signaling. The results of these studies should help elucidate the mechanism of WASP function in T cell activation and will have important implications for our understanding of normal immunity to infection and for immunologic diseases that include immunodeficiency diseases, cancer, and autoimmunity and allergic diseases.